Marrow stem cells have recently been shown capable of differentiating into a number of nonhematopoietic cell types in different tissues including cardiac myocytes, hepatocytes, neuronal cells, epithelial cells (see project #1), vascular endothelial cells, cartilage and bone. Conversion of green-fluorescent protein (GFP) positive marrow cells to skeletal muscle cells has also been demonstrated and is the focus of this project. We have determined that donor GFP+ marrow cells have converted to muscle cells by colocalizing GFP and desmin in morphologically characteristic muscle fibers. We have found increasing levels of marrow conversion or transdifferentiation to skeletal muscle cells by altering the specifics of the transplant regimen including cell number, timing of transplant and mode of cell delivery (i.e. local injection vs systemic infusion or mobilization of transplanted cells). We have also found that the number of conversion events changes with different mobilization regimens and that subsets of marrow cells, i.e. Dexter culture adherent cells and especially lineage negative cells, give higher rates of conversion than unseparated marrow cells. The nature of the skeletal muscle injury, radiation or direct cardiotoxin injection, is also critical in determining the level of transdifferentiation of marrow to muscle cells seen in vivo. The relevance of these studies to possible clinical application depends upon the "robustness" of the conversions. In settings without injury or in some of our experimental models the levels of conversion have been quite low, at the 0.01% level. However, in preliminary studies using a cardiotoxin muscle injury in previously transplanted mice, combined with radiation and direct injection of different populations of marrow cells we have obtained conversion rates up to 12%, i.e. 12% of the muscle fibers were GFP+ skeletal muscle cells. This, to our knowledge, is the highest rate of marrow conversion to muscle cells in the published literature, and can be considered as relatively "robust" and a major step to clinically significant "robustness". In these same studies, we have also observed, for the first time, colonies of GFP+ muscle cells in the anterior tibialis muscle. Our present proposal is to continue to evaluate the specifics of muscle injury which will lead to high-level conversion of marrow to muscle and to explore which particular cell type can give rise to muscle at the highest frequency, the timing of transplant suitable for such conversions and the number of cells necessary to obtain significant muscle conversion. We will also test the effect of different "muscle active" cytokines (HGF, LIF, FGF6, and IGF1 ) on the differentiation of marrow to muscle cells. We will then apply our optimized system to relevant models of muscular dystrophy in mice. We are also particularly interested in the possibility that work in project three on siRNA may open up possibilities blocking specific hematopoietic stem cell differentiation pathways and diverting them to a myogneic pathway. Thus we plan to work with project 1 on PU. 1 blockade and its effect of marrow to muscle conversions. As this work proceeds we would of course evaluate other transcriptional regulatory blockades. The knowledge gained in this grant can be applied for the development of clinical protocols for treatment of muscular dystrophy and other genetic diseases.